Regulation of cardiac patterning by the BAF complex subunit Smarcc1a

项目概要 心脏模式是在胚胎内创建具有独特功能的不同区域的重要过程 心。随着心脏的发育，它从简单的线性心管转变为具有心房的图案结构 心室和房室管（AVC）在它们的交界处。这些的形成 形态学差异伴随着基因表达模式的动态变化：处于 首先在整个心管中广泛表达，后来限制在心室或 AVC 中。尽管 虽然 AVC 和腔室图案化的功能重要性，但我们尚未完全了解其分子机制 促进特定基因限制在任一区域。 为了解决这个悬而未决的问题，我们的实验室对斑马鱼进行了基因筛查以寻找突变 破坏 AVC 或心室的形态。在之前的屏幕中，我们发现了一个突变体 AVC 和心室形态发生严重缺陷，导致心脏相对线性，没有 心房和心室边界处的 AVC 形态不同。另外，变异之心 未能对 AVC 的基因表达进行适当的细化。定位克隆表明 致病突变破坏了斑马鱼 smarcc1a 基因，编码 SWI/SNF 型 ATP 依赖性染色质 与哺乳动物 BAF155 同源的重塑复杂亚基。这些结果提出了一种新颖的模型，其中 含有 Smarcc1a 的 BAF 复合物进行的染色质重塑完善了基因表达模式， 促进 AVC 和商会身份之间的区别。 为了测试这个模型，我将首先确定 Smarcc1a 何时何地发挥作用，限制 AVC 的基因表达。 具体来说，我将检查 smarcc1a 对基因表达区域模式的影响程度， 确定 smarcc1a 突变体中首次出现基因表达缺陷的时间，并利用转基因策略 smarcc1a 突变体的组织特异性拯救。此外，我将确定作用于下游的效应基因 Smarcc1a 驱动心脏模式。使用功能丧失、功能获得和上位分析，我将测试 smarcc1a 是否通过影响 tbx2b 表达的抑制来控制心脏模式。我也会 使用转录组学方法来识别更广泛的受 smarcc1a 调控的候选基因， 我将测试最佳候选者是否是执行心脏模式的关键效应器。 总之，这些研究很可能揭示 smarcc1a 在调节区分机制的新作用 从相邻心室的特征中得出 AVC 的特征。 smarcc1a 的这个角色代表 BAF 染色质重塑复合物在心脏模式形成过程中的功能此前未被认识到。因此，这个 这项工作有可能产生更广泛的生物医学影响，因为它可以增强我们对如何 编码染色质重塑复合物成分的基因突变可能与染色质重塑复合物的起源有关 先天性心脏病。

PROJECT SUMMARY Cardiac patterning is an essential process that creates distinct regions with unique functions within the embryonic heart. As the heart develops, it transforms from a simple linear heart tube into a patterned structure with atrial and ventricular chambers and the atrioventricular canal (AVC) at their junction. The formation of these morphological distinctions is accompanied by dynamic changes in gene expression patterns: genes that were at first broadly expressed throughout the heart tube become restricted to either the chambers or the AVC. Despite the functional importance of AVC and chamber patterning, we do not fully understand the molecular mechanisms that promote the restriction of specific genes to either region. To address this open question, our laboratory has carried out genetic screens in zebrafish in search of mutations that disrupt the morphology of the AVC or the cardiac chambers. In a previous screen, we identified a mutant with severe defects in AVC and chamber morphogenesis, resulting in a relatively linear heart without a morphologically distinct AVC at the boundary between the atrium and the ventricle. In addition, the mutant heart fails to exhibit the appropriate refinement of gene expression to the AVC. Positional cloning revealed that the causative mutation disrupts the zebrafish smarcc1a gene, encoding a SWI/SNF-type ATP-dependent chromatin remodeling complex subunit homologous to mammalian BAF155. These results suggest a novel model in which chromatin remodeling by Smarcc1a-containing BAF complexes refines the gene expression patterns that promote the distinction between AVC and chamber identities. To test this model, I will first determine when and where Smarcc1a acts to restrict gene expression to the AVC. Specifically, I will examine the extent to which smarcc1a influences regional patterns of gene expression, determine when gene expression defects first emerge in smarcc1a mutants, and utilize transgenic strategies for tissue-specific rescue of smarcc1a mutants. Additionally, I will identify effector genes that act downstream of Smarcc1a to drive cardiac patterning. Using loss-of-function, gain-of-function, and epistasis analysis, I will test whether smarcc1a controls cardiac patterning through its influence on repression of tbx2b expression. I will also use transcriptomic approaches to identify a broader roster of candidate genes that are regulated by smarcc1a, and I will test whether top candidates are key effectors in executing cardiac patterning. Together, these studies are likely to reveal a novel role of smarcc1a in regulating the mechanisms that distinguish the traits of the AVC from the traits of the adjacent cardiac chambers. This role of smarcc1a would represent a previously unappreciated function for BAF chromatin remodeling complexes during cardiac patterning. Thus, this work has the potential to have a broader biomedical impact, as it may enhance our understanding of how mutations in genes encoding components of chromatin remodeling complexes could relate to the origins of congenital heart disease.